Optimal cell function requires a fine balance between the synthesis and degradation of biomolecules. Autophagy is the process by which cells degrade and recycle their own components, helping to clean up and maintain the cell’s internal environment and ensure the smooth functioning of cellular processes. Autophagy is strongly induced when cells are subjected to stresses like nutrient deprivation, acting under such conditions to supply nutrients through its breakdown of unneeded cellular material.
Autophagy substrates are delivered to vacuoles in yeast or lysosomes in mammals for degradation by double-membrane vesicles called “autophagosomes”. While autophagy was originally considered a non-selective process that isolates substrates in the cytoplasm of the cell in a random manner, studies have reported that certain cellular components, such as a subset of proteins and damaged or superfluous cell organelles, are isolated in a selective manner. In contrast to this well-established targeting of organelles and proteins by autophagy, the question of whether RNAs are subjected to autophagy and if they are selectively degraded has remained unanswered.
In their latest study, which was published in Nature Communications, researchers from the Tokyo Tech and RIKEN conducted a detailed analysis of the preferential degradation by autophagy of messenger RNAs (mRNAs), which contain the information required to make cellular protein and bind ribosomes for protein synthesis. Corresponding author Prof. Yoshinori Ohsumi of the Tokyo Tech, who was awarded the 2016 Nobel Prize in Physiology or Medicine for his pioneering work in the field of autophagy, explained the group’s findings, stating “We have previously shown that RNA delivered to the vacuole via autophagy in yeast cells, where it is degraded by vacuolar nucleases. The question of whether RNA degradation by autophagy occurs preferentially, however, remains unaddressed. This difficult to address question was the starting point of this project.”
As RNAs that accumulate in the vacuole are enzymatically degraded by the nuclease Rny1, they first constructed a yeast strain lacking this enzyme. Using this strain, they were able to isolate and identify RNAs that accumulated in the vacuole. Next, they used the drug rapamycin, which is known to induce autophagy, to assess unique features of mRNA species delivered to the vacuole in Rny1-deficient cells when autophagy is induced. Critically, they discovered that autophagy-mediated mRNA delivery to vacuoles is selective, not random, in nature.
The researchers then characterized the different mRNA species by conducting a broad analysis of the types of mRNAs in these cells, identifying ‘vacuole-enriched’ and ‘vacuole-depleted’ mRNAs. Interestingly, housekeeping mRNAs, such as those encoding proteins involved in amino acid biosynthesis, were most likely to be delivered to vacuoles. In contrast, mRNAs required for the synthesis of proteins with regulatory functions, such as protein kinases, were predominantly detected in the vacuole-depleted mRNA fraction.
Furthermore, they demonstrated that mRNAs undergoing translation are delivered to the vacuole, which is suggested to be a translation-dependent process. Moreover, persistent ribosome-mRNA association upon rapamycin treatment was found to be a key determinant of vacuolar mRNA delivery during autophagy-mediated degradation.
Dr. Makino and Prof. Ohsumi highlighted the importance of autophagy in gene regulation, remarking, “Our findings suggest that autophagy regulates mRNA degradation at the translation step, thereby enabling a rapid and sensitive switch from ribosome-associated mRNAs to expression of mRNAs that are essential for an effective response to stress. Preferential degradation of ribosome-mRNAs by autophagy is therefore very likely to determine the fate of individual mRNAs as cells adapt to new conditions.”
Reference: Makino, S., Kawamata, T., Iwasaki, S. et al. Selectivity of mRNA degradation by autophagy in yeast. Nat Commun 12, 2316 (2021). https://doi.org/10.1038/s41467-021-22574-6
Researchers report that pancreatic cancer tumors use multiple mechanisms to avoid starvation, suggesting a new target for treating a very difficult and deadly disease
In new findings published online March 18, 2021 in the journal Cancer Cell, an international team of researchers, led by scientists at University of California San Diego School of Medicine and Moores Cancer Center, describe how pancreatic cancer cells use an alternative method to find necessary nutrients, defying current therapies, to help them grow and spread.
Pancreatic cancer accounts for roughly 3 percent of all cancers in the United States, but it is among the most aggressive and deadly, resulting in 7 percent of all cancer deaths annually. Pancreatic cancer is especially deadly once it metastasizes, with the number of people who are alive five years later declining from 37 percent to just 3 percent.
All cancer cells require a constant supply of nutrients. Some types of cancer achieve this by creating their own vascular networks to pull in nutrients from the host’s blood supply. But other cancers, notably pancreatic ductal adenocarcinoma, are surrounded by a thick layer of connective tissue and extracellular molecules (the so-called tumor stroma) that act not just as a sort of a dividing line between malignant cells and normal host tissues, but also as a hindrance to cancer cells obtaining sufficient resources, including blood supply.
As a result, pancreatic and other nutritionally stressed cancers employ a number of adaptive mechanisms to avoid death by starvation, a risk particularly high in rapidly growing tumors. One such mechanism is autophagy or self-eating. Autophagy allows nutritionally stressed cancers to digest intracellular proteins, especially denatured or damaged proteins, and use the liberated amino acid building blocks as an energy source to fuel their metabolism.
Past research indicating autophagy is elevated in pancreatic cancer gave rise to the idea that inhibiting self-eating might be used to starve tumors. Yet, multiple clinical trials using compounds that inhibit autophagic protein degradation combined with traditional chemotherapy, did not produce any added therapeutic benefit compared to chemotherapy alone, said Michael Karin, PhD, Distinguished Professor of Pharmacology and Pathology at UC San Diego School of Medicine.
In the new study, Hua Su, PhD, a postdoctoral fellow in Karin’s lab and first author of the study, and collaborators investigated why pancreatic cancers survive autophagy and, in fact, appear to thrive. They found that inhibition of autophagy resulted in rapid upregulation or increased activity of a different nutrient procurement pathway called macropinocytosis, derived from the Greek for “large drinking or gulping.”
Macropinocytosis enables autophagy-compromised and nutritionally stressed cancer cells to take up exogenous proteins (found outside the cell), digest them and use their amino acids for energy generation. “This explains why autophagy inhibitors fail to starve pancreatic cancer and cannot induce its regression,” said Su. “Once autophagy is inhibited, cancer cells simply resort to a different mechanism to feed themselves.”
In experiments using mouse cancer models and human pancreatic cancers grown in mice, Su and colleagues found that a combination of autophagy and macropinocytosis inhibitors resulted in rapid and nearly complete tumor regression.
“These results provide another example of the plastic nature of pancreatic cancer metabolism,” said senior author Karin. “It also shows that combined inhibition of the two major nutrient procurement pathways can result in a successful blockade of energy supply resulting in tumor starvation and consequent shrinkage.”
Study co-author Andrew Lowy, MD, chief of the Division of Surgical Oncology at Moores Cancer Center at UC San Diego Health and a professor of surgery at UC San Diego School of Medicine, said the new data demonstrate the promise of targeting tumor metabolism as a treatment strategy and that success will likely require combining multiple agents for multiple targets.
“I believe that these findings are exciting and support the idea that we will make significant impact against this very difficult disease in the near-future,” Lowy said.
Co-authors include: Fei Yang, Rao Fu, Xiaohong Pu and Beicheng Sun, Nanjing University Medical School; Xin Li and Yinling Hu, National Cancer Institute; Randall French, Evangeline Mose, Brittney Trinh, Junlai Liu, Laura Antonucci, Yuan Liu, Avi Kumar and Christian M. Metallo, UC San Diego; Jelena Todoric, UC San Diego and Medical University of Vienna; Maria Diaz-Meco and Jorge Moscat, Weill Cornell Medicine.
Funding for this research came, in part, from Padres Pedal the Cause/C3 (PPTC2018), the UC Pancreatic Cancer Consortium, the National Institutes of Health (R01CA211794, R37AI043477, P01DK098108, R01CA155630, R03CA223717, R01CA234245, R01CA218254, R01DK108743), the Youth Program of the National Natural Science Foundation of China (81802757, 82002931) and the National Key Research and Development Program of China (2016YFC0905900).
Featured image: Pancreatic cancer cells (blue) growing as a sphere encased in membranes (red). Photo credit: National Cancer Institute
Reference: Hua Su, Rao Fu, Fei Yang et al., “Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis”, Cancer Cell, 2021. DOI: https://doi.org/10.1016/j.ccell.2021.02.016
Autophagy is an intracellular degradation process of cytosolic materials and damaged organelles. Researchers at Ubiquitin Project of TMIMS have been studying the molecular mechanism of mitophagy, the selective autophagy process to eliminate damaged mitochondria. PINK1 (a serine/threonine kinase) and Parkin (a ubiquitin ligating enzyme: E3) work together to ubiquitylate the outer membrane proteins of damaged mitochondria, then ubiquitin chains are recognized as signals for autophagy degradation. Dysfunction of mitophagy causes a decrease in mitochondrial quality with overproduction of ROS, and is linked to neurodegenerative diseases like Parkinson’s disease.
In Autophagy machinery, cellular components targeted for degradation are engulfed by phosphatidylinositol-3-phosphate (PI3P)-rich membranes. Membranes are elongated and enclosed to form autophagosomes, which then fuse with lysosomes to degrade the cargo inside. Many proteins function in autophagy machinery and they were initially identified by genetic screens in the budding yeast Saccharomyces cerevisiae, and Caenorhabditis elegans. Essential autophagy proteins are evolutionarily conserved from yeast to humans. However, in mammals, there should be unidentified autophagic proteins, and accessory components, whose single gene deletions only manifest as mild defects in autophagy activity, might be missed by these types of genetic screens.
In this study, by immunoprecipitating WIPI1, the well-known autophagy protein, upon Parkin-mediated mitophagy-inducing conditions, researchers identified human BCAS3 (Breast Carcinoma Amplified Sequence 3) and C16orf70 (chromosome 16 open reading frame 70) as novel autophagic proteins.
While BCAS3 and C16orf70 are dispersed throughout the cytosol under normal condition, they accumulated around the damaged mitochondria after mitophagy induction. They also formed puncta in the cytosol in response to amino-acid starvation, which suggests that BCAS3 and C16orf70 are recruited to the autophagosome in both non-selective and selective autophagy. Researchers then found that BCAS3 and C16orf70 interact each other, and this interaction is required for their accumulation on the autophagosome formation site.
Autophagy efficiencies in response to mitochondrial damage and amino-acid starvation were not affected by BCAS3 and/or C16orf70 gene deletions at least in cultured cells. On the other hand, overexpression of the BCAS3-C16orf70 complex impairs the assembly of several autophagy core proteins. These findings demonstrate important accessory functions of BCAS3 and C16orf70 in autophagy machinery.
Furthermore, in silico structural modeling of BCAS3 followed by mutational analyses in immunocytochemistry and in vitro phosphoinositide-binding assays indicate that BCAS3 directly binds phosphatidylinositol-3-phosphate on the autophagosome membranes.
This work was conducted by researchers in TMIMS, The University of Tokushima, and National Institute of Advanced Industrial Science and Technology (AIST), Japan.
This work was supported by JSPS KAKENHI Grant JP17J03737, JP18H05500, JP18K06237, JP18KK0229, JP19H04966, JP20K06628, JP18H02443, JP19H05712, JP19H00997, 16K21680, 18K11543, the Chieko Iwanaga Fund for Parkinson’s Disease Research, the Takeda Science Foundation and Joint Usage and Joint Research Programs, the Institute of Advanced Medical Sciences, Tokushima University and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant numbers JP19am0101114.
Reference: Waka Kojima, Koji Yamano, Hidetaka Kosako, Kenichiro Imai, Reika Kikuchi, Keiji Tanaka & Noriyuki Matsuda (2021) Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy, Autophagy, DOI: 10.1080/15548627.2021.1874133
UC researchers found that stopping cell recycling could help treat aggressive breast cancer
Recycling cans and bottles is a good practice. It helps keep the planet clean.
The same is true for recycling within cells in the body. Each cell has a way of cleaning out waste in order to regenerate newer, healthier cells. This “cell recycling” is called autophagy.
Targeting and changing this process has been linked to helping control or diminish certain cancers. Now, University of Cincinnati researchers have shown that completely halting this process in a very aggressive form of breast cancer may improve outcomes for patients one day.
These results are published in the Feb. 8 print edition of the journal Developmental Cell.
First image (left): Green marks the HER2 protein on the tumor cell surface, which drives breast cancer development and growth of tumor cells. Image two and three: After blocking the cell recycling process, known as autophagy, by eliminating the genes that cause it, HER2 is prevented from reaching the cell surface and accumulates in fluid-filled pouches, called vesicles (white arrows) and is eventually released from cells, stopping tumor cell growth. Provided by Guan’s lab
“Autophagy is sort of like cell cannibalism,” says corresponding author Jun-Lin Guan, PhD, Francis Brunning Professor and Chair of UC’s Department of Cancer Biology. “They eat the nasty components of themselves and come out strong and undamaged; however, we do not want cancer cells doing this to create stronger, healthier versions of themselves. Previous studies found that disabling this process slowed down the growth of another type of breast cancer, but it was unknown whether blocking autophagy could be beneficial for a particularly aggressive type of breast cancer, known as HER2-positive breast cancer.”
This type of breast cancer grows rapidly, and while there are effective treatments, unfortunately, these particular cancer cells find a way to become resistant to therapy, leading to relapse and a higher death rate in patients.
Researchers in this study used animal models to show that blocking autophagy eliminated the development and growth of this type of breast cancer “even to a greater extent than our previous studies in other types of breast cancer,” says Guan, also a member of the UC Cancer Center.
Researchers in Jun-Lin Guan’s lab have found that eliminating “cell recycling” in certain cancer cells could help in treating a very aggressive form of breast cancer. Photo/Colleen Kelley
He adds that researchers also uncovered that by blocking this activity, they were able to impact the other activities and mechanisms within the cancer cells completely, changing their roles and reactions.
“It altered trafficking patterns of the HER2 protein after it is produced by the cancer cells,” he continues. “Instead of being put in its ‘normal’ location on the cell surface to cause cancer development, it is incorporated into some small fluid-filled pouches, known as vesicles, and secreted out of the tumor cells.”
Guan says these findings are particularly important as they show a completely different way to potentially treat this type of breast cancer and may work as a combination therapy with current treatments to prevent resistance and relapse.
This study really shows the value of basic research in beating cancer in the future. Breakthroughs, like this one, are sometimes made from curiosity-driven research that result in surprising findings that could one day help people.
— Jun-Lin Guan, PhD
“It would be harder for the cancer cells to develop ways to evade two different ways to be blocked,” he adds. “Future clinical studies will be needed to validate the treatment in human patients. Also, the HER2 protein plays a role in several other cancers including lung, gastric [stomach] and prostate cancers, so future studies will need to examine whether this new mechanism may also be beneficial in treating those cancers as well.
“This study really shows the value of basic research in beating cancer in the future. Breakthroughs, like this one, are sometimes made from curiosity-driven research that result in surprising findings that could one day help people.”
Findings that fuel future research
Breast cancer cells. Courtesy of the National Cancer Institute.
Lead author on the study Mingang Hao, PhD, who is a postdoctoral fellow in Guan’s lab, says he was handling two separate cancer research projects at the same time, but this study inspired findings for the other, which also involved vesicles or “bubbles” in cancer spread.
“Cancer research has so many intricate twists and turns, but so much of it can be interconnected, even in tiny ways,” Hao says. “Working with the teams at UC has shown me some really innovative ways to tackle this disease, and I’m able to apply things I’m learning in one lab to research in another, to ultimately help find solutions for this terrible disease.”
Co-author Kevin Turner, MD, a resident in the Department of Surgery at UC, says his work with this science helps him understand more about cancer development and spread to better treat patients.
“As a surgical resident planning to pursue a career in surgical oncology, having the opportunity to work in a science lab with Dr. Guan and his team has allowed me to develop a deeper understanding of the workings of a disease I have seen in my patients,” he says. “I hope to continue studies on this as we work toward clinical trials and applying it in patients.”
This research was funded by the National Institutes of Health (R01 CA211066; R01 HL073394; R01 NS094144). Researchers cite no conflict of interest.
Featured photo of 3D breast cancer cell courtesy of the National Cancer Institute.
Reference: Mingang Hao, Syn Kok Yeo, Kevin Turner, Alexis Harold, Yongguang Yang, Xiaoting Zhang, Jun-Lin Guan, Autophagy Blockade Limits HER2+ Breast Cancer Tumorigenesis by Perturbing HER2 Trafficking and Promoting Release Via Small Extracellular Vesicles, Developmental Cell, Volume 56, Issue 3, 2021, Pages 341-355.e5, ISSN 1534-5807, https://doi.org/10.1016/j.devcel.2020.12.016. (https://www.sciencedirect.com/science/article/pii/S1534580720310224)
A new study shows that the release of stored iron in heart cells may contribute to heart failure, suggesting potential new approaches to treatment.
A process that releases iron in response to stress may contribute to heart failure, and blocking this process could be a way of protecting the heart, suggests a study in mice published today in eLife.
People with heart failure often have an iron deficiency, leading some scientists to suspect that problems with iron processing in the body may play a role in this condition. The study explains one way that iron processing may contribute to heart failure and suggests potential treatment approaches to protect the heart.
“Iron is essential for many processes in the body including oxygen transport, but too much iron can lead to a build-up of unstable oxygen molecules that can kill cells,” says first author Jumpei Ito, who was a Research Associate at the School of Cardiovascular Medicine and Sciences, King’s College London, UK, at the time the study was carried out, and is now a visiting scientist based at Osaka Medical College, Japan. “We already knew that iron metabolism undergoes changes in heart failure, but it was unclear whether these changes are helpful or harmful.”
To learn more about the role of iron metabolism in heart failure, Ito and colleagues studied mice lacking a protein called the nuclear receptor coactivator 4 (NCOA4), which is necessary to release iron stored in cells when the body’s iron levels are low. They found that these mice developed less severe changes associated with heart failure compared to mice with NCOA4. Specifically, the NCOA4-deficient mice did not develop excessive levels of iron or a build-up of unstable oxygen molecules that can lead to cell death in heart failure.
A compound called ferrostatin-1 inhibits the release of stored iron and reduces the accumulation of unstable oxygen molecules. Further experiments by the team showed that treating mice with NCOA4 with ferrostatin-1 can reduce the amount of cell death in heart failure. “Our results suggest that the release of iron can be detrimental to the heart,” Ito says. “It can lead to unstable oxygen levels, death in heart cells and ultimately heart failure.”
More studies are now needed to understand each step in the process that releases iron and to test whether inhibiting this process could be beneficial to people with heart failure.
“Patients with heart failure who are iron deficient are currently treated with iron supplements, which previous studies have shown reduces their symptoms,” adds senior author Kinya Otsu, the British Heart Foundation Professor of Cardiology at King’s College London. “While our work does not contradict those studies, it does suggest that reducing iron-dependent cell death in the heart could be a potential new treatment strategy for patients.”
Reference: Jumpei Ito et al., “Iron derived from autophagy-mediated ferritin degradation induces cardiomyocyte death and heart failure in mice”, eLife, 2021. DOI: 10.7554/eLife.62174
Autophagy is a fundamental cellular process by which cells capture and degrade their own dysfunctional or superfluous components for degradation and recycling. Recent research has revealed that phase separated droplets have a range of important functions in cells. An international collaboration between German, Norwegian, and Japanese researchers has unravelled the mechanisms underpinning both how these droplets are captured through autophagy, as well as how droplets can serve as a platform from which structures facilitating cytosolic autophagy arise.
Autophagy[1], a critical intracellular degradation pathway that plays a key role in human health, has attracted the attention of cell biologists for decades, culminating in the award of the 2016 Nobel Prize in Physiology or Medicine to Tokyo Institute of Technology (Tokyo Tech) Specially Appointed Professor Yoshinori Ohsumi in 2016 for his work uncovering the mechanisms of this process. Recently, the autophagy has been observed to degrade fluid droplets[2], which are formed by phase separation and have been identified as important structural components of cells in rapidly progressing research. But how this ‘eating’ of fluid droplets occurs is unknown.
This simple but important question prompted Dr Roland Knorr at the University of Tokyo to assemble an international team of researchers from Göttingen (Germany), Oslo (Norway), and Tokyo (Japan), including Dr Alexander I. May from the Institute of Innovate Research at Tokyo Tech. This group set out to understand the biological process of autophagosomal droplet sequestration, discovering that an intricate physical mechanism underlies the relationship between autophagy and droplets. Their results, published in this week’s issue of Nature, represent a major breakthrough in our understanding of how autophagy captures cellular material and how droplets are degraded in cells. These findings promise to inform therapeutic studies targeting autophagy and the abnormal accumulation of droplet materials observed in neurodegenerative and other diseases.
One bite at a time
In the first step of autophagy, the isolation membrane, a key functional structure of autophagy made up of a double-layered lipid membrane shaped somewhat like a flattened tennis ball, grows in size, bends to form a cup-like shape and ultimately forms a spherical structure known as the autophagosome. Autophagosomes capture cytosolic and other cellular material such as droplets, isolating this cargo from the rest of the cytosol, following which the cargo is broken down and its building blocks recycled by the cell. The researchers focused on the isolation of droplets, which they found can be understood in terms of surprisingly simple and fundamental physical principles.
Droplets are spherical due to the effect of surface tension, which acts to minimize a droplet’s surface area. How strongly a droplet can resist deformations from a spherical shape is defined by the droplet’s surface tension[3], the value of which reflects how strongly the droplet and the surrounding cytosol repel each other. Critically, lipid membranes are able to sit at the interface between the droplet and cytosolic fluids, a phenomenon known as wetting. Wetting depends on how strong a membrane favours interaction with the droplet and the cytosol, as well as the droplet surface tension.
The researchers developed a theoretical model that accounts for these physical forces to explain how autophagy membranes interact with and capture droplets. They found that the shape of the droplet-isolation membrane pair is governed by a competition between the droplet’s resistance to deform and the tendency of the isolation membrane to bend. Dr. May explains how physical forces determine the outcome of the droplet-isolation membrane interactions: “During the initial phase of autophagy, isolation membranes on droplets are small, which means they only have a weak tendency to bend. As the membrane area grows, however, these membranes become more likely to bend – the bending energy increases. The droplet’s surface tension defines its resistance to deformation, and if the surface tension is low enough a critical point can be reached where the bending energy of the isolation overcomes the droplet’s surface tension. In this case, a piece of the droplet is ‘bitten off’ and captured within an autophagosome. If this critical point is never reached and the surface tension of the droplet ‘wins’ this competition by overcoming the membrane bending energy, the isolation membrane will continue to grow along the droplet surface, eventually engulfing the entire droplet. Droplet autophagy can therefore be thought of as a sort of tug-of-war between the droplet’s surface tension and the isolation membrane’s bending energy.”
With the model predicting this trade-off between ‘piecemeal’ and ‘complete’ autophagy, the team set out to confirm these findings in living cells. The researchers used a cutting-edge combination of fluorescence and electron microscopy to follow droplet compartments that enrich a protein called p62 or SQSTM1[4]. As predicted by modelling of low surface tension droplet conditions, the localisation of small isolation membranes to the droplet surface was followed by the ‘biting off’ of pieces of droplet. But the team needed to develop an innovative means of controlling droplet surface tension to confirm the influence of droplet properties on sequestration.
Autophagy on demand
To address this question, the researchers devised a minimal synthetic experimental system that eliminates the complexity of the intracellular environment. Using this approach, they observed the self-assembly of isolation membrane-like structures from pre-existing membranes on the surface of droplets with high surface tension. The tuneable nature of this experimental setup allowed the researchers to decrease droplet surface tension, thereby testing what effect this has on droplet capture. As predicted by the model, they observed that flattened isolation membranes transform via an intermediate cup-like shape into an autophagosome-like structure, thereby taking a bite from the droplet. Together, these results confirm the veracity of the model and demonstrate that wetting is the physical mechanism governing autophagosome formation at droplets.
These results indicate that biologists are still exploring only the tip of the iceberg when it comes to the significance of phase separation in autophagy. Intriguingly, another study published in Nature last year that was co-authored by Dr. Ohsumi, Dr. Knorr and Dr. May showed that the site of autophagosome formation in yeast cells is in fact a fluid droplet that is never captured. Dr. Knorr remarks: “I was very fascinated to discover droplets being a novel key autophagy structure. Now, we wanted to understand the mechanism behind our observation that some types of droplets are degraded by autophagosomes, such as p62, but others not, including the site of autophagosome formation.”
Switching things up
The simple competition between isolation membrane bending and droplet surface tension described above assumes that the properties of the isolation membrane aren’t altered when it sticks to the droplet surface. This is unlikely as each side of the isolation membrane wets two very different fluids during droplet autophagy: the droplet or the cytosol. The team expanded on their model to account for this, finding that such wetting-derived intrinsic asymmetry of isolation membranes determines bending direction and thereby the material captured for degradation: either the droplet via the piecemeal pathway, or the cytosol through the growth of the isolation membrane away from the droplet. The upshot of this is that the particular combination of isolation membranes, droplet properties and cytosolic state combine to specify the droplet as a target for autophagy or, counterintuitively, as a platform that enables autophagy of the surrounding cytosol.
To test this, the researchers modified the p62 protein to lack a specific motif that is known to interact with the proteins in the isolation membrane, thereby weakening the isolation membrane-droplet association. This manipulation had a radical effect: while isolation membranes were initially observed to grow along p62 droplets in wild-type (unmodified) cells, they instead bent to capture cytosol, leaving the droplet completely intact. Tiny changes in droplet properties therefore have critical implications for the mode of autophagy in living cells, specifying piecemeal or complete enclosure of droplets, and even the capture of cytosolic material.
Elucidation of the underlying physical rationale that enables this switch provides an entirely new perspective in our understanding of the mechanism of autophagy, as well as the role of droplets and physical principles such as wetting in cells. This understanding lays the groundwork for a host of new studies on the implications of physical forces in cell biology, as well as providing new clues that will help understand how autophagy is involved in diseases that are not easily treated, such as neurodegenerative diseases and cancer.
Footnotes
[1] Autophagy is an important intracellular degradation pathway that has been linked to many important processes in healthy cells, such as ensuring reliable supply of metabolite concentrations, starvation responses and maintenance of the cell’s population of organelles. The disruption of autophagy is associated with diseases including infections, neurodegenerative diseases and cancer. Gaining a detailed understanding of how autophagy occurs in cells therefore promises novel means of addressing human diseases.
[2] Droplets, also known as ‘membrane-less organelles,’ are condensates of proteins that form by phase separation-like processes. These structures lack a limiting membrane, therefore behaving as a dynamic yet discrete fluid in the cytosol (the cell’s interior solution), akin to droplets of oil in water. Droplets have recently attracted a lot of research attention due to their increasingly recognised physiological importance, but the question of how they are degraded or dissociate is only poorly understood.
[3] Surface tension is a force that causes the surface of liquids, including fluid droplets in cells, to minimise their surface area. In physical terms, a liquid’s surface tension is characterised by a value that depends on the properties of the liquid and its surrounding material.
[4] p62/SQSTM1 is a protein that is degraded by autophagy. It acts as an adaptor protein by binding to other cellular proteins through a region known as the LC3-interacting region (LIR), thereby allowing for the degradation of these proteins by autophagy. p62/SQSTM1 is known to form droplets in cells that can mature into relatively inert aggregations associated with neurodegenerative disease.
Reference: Jaime Agudo-Canalejo, Sebastian W. Schultz, Haruka Chino, Simona M. Migliano, Chieko Saito, Ikuko Koyama-Honda, Harald Stenmark, Andreas Brech, Alexander I. May, Noboru Mizushima & Roland L. Knorr, “Wetting regulates autophagy of phase- separated compartments and the cytosol”, Nature, 2021. DOI :10.1038/s41586-020-2992-3https://www.nature.com/articles/s41586-020-2992-3
Proteins provide important raw materials for the self-renewal of cells in the gastrointestinal tract. Cell self-renewal is inseparable from the coordination between apoptosis and autophagy.
However, there are few reports on the relationship between different nitrogen sources and apoptosis/autophagy.
Researchers from the Institute of Subtropical Agriculture (ISA) of the Chinese Academy of Sciences and Shenzhen University compared the effects of different nitrogen sources on apoptosis and autophagy of rumen epithelial cells in goats. They found that nonprotein nitrogen and protein nitrogen regulate cell self-renewal in different manners.
The study has been published in Animals on Nov. 9.
The researchers found that ammonium chloride (NH4Cl) induced cell apoptosis by causing mitochondrial dysfunction. The levels of autophagy-related proteins and the numbers of autophagosomes were higher in NH4Cl-treated cells than in methionine (Met)-treated cells.
The results showed that endoplasmic reticulum (ER) stress played a critical role in the crosstalk between apoptosis and autophagy induced by NH4Cl and Met. And autophagy had a less obvious ameliorative effect on ruminal epithelial cell apoptosis after treatment with protein nitrogen than after treatment with nonprotein nitrogen. This maybe because protein nitrogen better suited than nonprotein nitrogen for protein synthesis and promoted the resistance of cells to AA deficiency stress.
Reference: Kong, Zhiwei; Zhou, Chuanshe; Kang, Jinhe; Tan, Zhiliang. 2020. “Comparison of the Effects of Nonprotein and Protein Nitrogen on Apoptosis and Autophagy of Rumen Epithelial Cells in Goats” Animals 10, no. 11: 2079. https://www.mdpi.com/2076-2615/10/11/2079#
Osteoarthritis is one of the most common problems associated with aging, and although there are therapies to treat the pain that results from the breakdown of the cartilage that cushions joints, there are no available therapies to modify the course of the disease.
However, working in a mouse model of the disorder, researchers at Washington University School of Medicine in St. Louis have found that a molecule previously linked to diabetes, cancer and muscle atrophy also seems to be involved in the development of osteoarthritis and may be a useful treatment target.
When the gene involved, FoxO1, is knocked out in mice, the animals develop osteoarthritis. But when the researchers increase the levels of the FoxO1 molecule in mice that are developing osteoarthritis, the animals exhibit less cartilage damage.
The study is available online in Proceedings of the National Academy of Sciences.
“Osteoarthritis, or joint degeneration, is a disease that affects more than 32 million people in the U.S. alone but that does not have a medical therapy to alter its progression,” said senior investigator Regis J. O’Keefe, MD, Ph.D., the Fred C. Reynolds Professor of Orthopedic Surgery and head of the Department of Orthopedic Surgery. “A better understanding of the fundamental processes involved in osteoarthritis and the degeneration of cartilage is required if we’re going to be more successful in treating this very common and very expensive disorder.”
O’Keefe said that commonly, people with osteoarthritis have suffered knee injuries that damaged the knee’s meniscus. Over time, arthritis then can develop in the joint.
“Unlike skin or bone or other organs that can regenerate in response to injury, cartilage has very little regenerative potential,” he said.
However, when the mice in these experiments had elevated levels of the FoxO1 molecule, osteoarthritis’s progress was slowed or even reversed. The researchers believe the molecule interferes with cartilage damage and the development of arthritis by enhancing a process called autophagy in the arthritic joint. Autophagy is the body’s way of clearing out damaged tissue. In these experiments, the researchers found that autophagy was disrupted in the mice with reduced levels of FoxO1 and that the process was enhanced in animals with higher levels of the molecule.
“In other words, maintaining a higher level of autophagy seemed to be beneficial to maintaining these cartilage cells and, thus, maintaining a healthy knee joint,” said co-corresponding author Jie Shen, assistant professor of orthopedic surgery.
O’Keefe said that raises the possibility of delivering FoxO1 to arthritic joints through nanotechnology as a way to regulate autophagy and keep joints healthier.
“In mice with injuries that typically progress to become osteoarthritis, the knee joints still appear normal about a week after injury,” O’Keefe explained. “But when we measure autophagy in the cartilage after injury to those same knee joints, although the joints themselves look fine, the autophagy process already is shut off. The injury completely turns it off, and once autophagy is off, the cartilage begins to degenerate.”
He said if FoxO1 can alter that process in people, protecting cartilage from damage as it does in mice, it eventually may be possible to prevent or delay millions of future knee and hip replacement surgeries.
References: Cuicui Wang et al. FoxO1 is a crucial mediator of TGF-β/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2017056117
Two-phase theory applies to diseases like Alzheimer’s, Parkinson’s, muscle atrophy.
Rice University biochemists Michael Stern and James McNew have studied how neurodegeneration kills cells. They’ve conducted countless experiments over more than a decade, and they’ve summarized all they’ve learned in a simple diagram they hope may change how doctors perceive and treat degenerative diseases as varied as Alzheimer’s, Parkinson’s and muscle atrophy.
Based on thousands of experiments over more than a decade, Rice University biochemists Michael Stern and James McNew believe degenerative diseases as varied as Alzheimer’s, Parkinson’s and muscle atrophy occur in two phases (green and red) marked by distinct alterations in the activity of the signaling pathway proteins TOR, AMPK, Foxo and JNK. In the early phase (left), autophagy is suppressed, which increases oxidative stress, eventually bringing about the late phase (right) in which autophagy is restored, causing rapid degeneration. (Illustration courtesy of M. Stern/Rice University)
In a study published this month in Molecular Psychiatry, McNew and Stern propose that degeneration, at the cellular level, occurs in two distinct phases that are marked by very different activities of protein signaling pathways that regulate basic cell functions.
“We would like clinicians and other researchers to understand that the two phases of degeneration represent distinct entities, with distinct alterations in signaling pathways that have distinct effects on disease pathology,” Stern said. “In other words, we think that patients need to be treated differently depending on which phase they are in.”
Stern and McNew’s diagram shows how the activity of key cell signaling proteins either increases or decreases at the onset of degeneration, ultimately bringing about oxidative stress. Oxidative stress then brings about the second phase of the condition, during which degeneration occurs, where the signaling proteins implicated in the first phase behave in a completely different way.
Because cells behave quite differently in the two phases, the research suggests patients in different phases of a disease may respond differently to the same treatment.
“The two phases of degeneration haven’t been previously recognized, so it hasn’t been understood, clinically, that you have two different populations of patients,” McNew said. “Today, they’re treated like one population, and we think this has confounded clinical trials and explains why some trials on Alzheimer’s have given variable and irreproducible effects. It would be like trying to treat all meningitis patients with antibiotics without realizing that there are two types of meningitis, one bacterial and one viral.”
Stern and McNew, professors of biochemistry and cell biology in Rice’s Department of BioSciences, became interested in the cellular processes of neurodegenerative disorders when they began studying hereditary spastic paraplegia (HSP) in the late 2000s. A rare disorder, HSP is marked by numbness and weakness in the legs and feet due to the progressive deterioration of neurons that connect the spine and lower leg.
These are some of the longest cells in the body, and starting with clues about structural defects that could cause them to degenerate, McNew and Stern used experiments on fruit flies to systematically piece together the biochemical domino effect that caused the neurons to progressively lose more and more function and eventually die. It had been thought that nerve damage could lead to muscle atrophy, but their studies found that muscle cells attached to the neurons started degenerating from the same type of biochemical cascade before the nerve cells died.
A key player in the cascade was a protein called TOR, a master regulator of cell growth and an essential protein for all higher order life from yeast to humans. TOR acts like a knob, dialing growth up or down to suit the conditions a cell is experiencing. In some conditions, high growth is warranted and beneficial, and in other situations growth needs to be dialed back so energy and resources can be conserved for daily chores, like the recycling or repair that take place during a process known as autophagy.
Some cancers highjack TOR to promote aggressive cell growth, and increased TOR activity has also been implicated in neurodegenerative disorders like Alzheimer’s and Parkinson’s diseases and in diseases marked by muscle atrophy. After compiling evidence about how TOR and several other signaling proteins behaved in neurodegeneration, McNew and Stern won a grant from the National Institute of Neurological Disorders and Stroke in 2018 for experiments to investigate signaling pathway changes that occur in the early stages of degeneration.
“At the time, we thought there might be a late phase during which degeneration actually occurs, but we didn’t propose any experiments to test that,” Stern said. “In the new paper, we’re explicit about the existence of a late phase. We propose mechanistically why degeneration occurs only during this phase, and cite abundant research in support.”
Stern said the two-phase process described in the study “is the basic engine that drives most or even all forms of degeneration forward. However, in addition, there are also inputs whose role is to specify how fast the engine turns over.”
To understand neurodegeneration, it’s critical to understand how those inputs work, he said. For example, insulin resistance plays a well-known role in driving Alzheimer’s disease, and in the study McNew and Stern describe how it does that by accelerating progression through the early phase.
“Similarly, our data suggests that decreases in synaptic transmission, as occurs in our HSP insect model, likewise triggers degeneration by accelerating progression through the early phase,” McNew said. “Our NIH grant was funded so that we could learn the mechanism by which that occurs.”
Now that they clearly understand that two phases of degeneration exist, Stern said he and McNew would like to carry out more experiments to see how the effects of specific genes on degeneration are altered when they are activated in the early and late phases.
“What we would like to do in the last two years of the grant is to obtain data to test some of the predictions we have made, which will help determine if the ideas we have presented are likely to be correct,” Stern said.
References: Michael Stern, James A. McNew. A transition to degeneration triggered by oxidative stress in degenerative disorders. Molecular Psychiatry, 2020; DOI: 10.1038/s41380-020-00943-9
